Methods and compositions for rapid purification of proteasomes and methods of use of components thereof

ABSTRACT

Disclosed are methods for rapidly and efficiently purifying proteasomes using fusion proteins having homology to ubiquitin. Also disclosed are methods for assessing aberrant cell growth rates utilizing fusion proteins have homology to ubiquitin and a signal producing moiety.

[0001] This application claims priority under 35 U.S.C. §119(e) fromU.S. Provisional Application, Serial No. 60/050,171 filed Jun. 19, 1997,the disclosure of which is incorporated by reference herein.

[0002] Pursuant to 35 U.S.C. §202(c) it is acknowledged that the U.S.Government has certain rights in the invention described herein, whichwas made in part with funds from the the National Institutes of Health(GM-52058).

FIELD OF THE INVENTION

[0003] This invention relates to the field of proteolytic degradation ofcellular proteins. More specifically, rapid and efficient methods forproteasome purification from various cell types are disclosed. Alsoprovided are novel methods for use of the proteasome components sopurified.

BACKGROUND OF THE INVENTION

[0004] Several publications are referenced in this application by authorname and year of publication in parentheses in order to more fullydescribe the state of the art to which this invention pertains. Fullcitations for these references are found at the end of thespecification. The disclosure of each of these publications isincorporated by reference herein.

[0005] The degradation of cellular proteins is necessary for thebiological well-being of all organisms. Regulators of cell growth anddevelopment, and components of the immune and cellular defensemechanisms are regulated by proteolysis. Membrane receptors andtranscription factors activated by cytokines, such as interleukins andinterferons, are regulated by protein degradation.

[0006] The major pathway of intracellular proteolysis involves theubiquitin/proteasome system. Ubiquitin, a 76 amino acid polypeptide, isthe most highly conserved protein in eukaryotic evolution. There areonly 3 amino acid differences between yeast and human ubiquitins.Extensive studies during the past decade have shown that the covalentattachment of ubiquitin to cellular proteins marks them for destruction.Substrates that are linked to ubiquitin are degraded by a multicatalyticprotease called the proteasome. During the past few years many targetsof the ubiquitin/proteasome system have been discovered and remarkablythey include a broad range of regulators of cell growth. Some of theproteins destroyed by the ubiquitin/proteasome system include cyclins,cyclin-dependent kinases (CDK's), NFκB, IκBα, cystic fibrosistransduction receptor, p53, ornithine decarboxylase (ODC), 7-membranespanning receptors, Cdc25 (phosphotyrosine phosphatase), Rb, Gα, c-Junand c-Fos.

[0007] The ubiquitin/proteasome pathway is also essential for thestress-response and for the generation of antigenic peptides in MHCClass I molecules. It is clear that defects in the functioning of theubiquitin/proteasome system can have severe consequences on biologicalhomeostasis. Indeed, mutations that affect the degradation of many ofthe proteins listed above have been associated with tumorigenesis.

[0008] The 26S-proteasome comprises two distinct sub-complexes. The corecomplex has a sedimentation velocity of 20S and contains a variety ofdegradative activities. The 20S core is highly conserved acrossevolutionary distance and consists of a barrel of 4 rings. Each ringcontains 7 subunits of either α class or β class. The rings are orientedso that two α-subunit-containing rings are on the outside, while twoβ-subunit containing rings are juxtaposed on the inside. Thus, the 20Score is identical at its two ends. The x-ray structure of thearchaebacterial proteasome has recently been resolved and was shown tocontain a narrow pore in each α ring, and a large central cavity formedby the β rings. Accordingly, the central cavity is not exposed to thecellular environment, thereby preventing non-specific degradation ofcellular proteins. Proteins targeted for degradation are first threadedthrough the narrow pores in the α rings before they gain access to thecentral catalytic cavity.

[0009] The second sub-complex, referred to as the 19S-regulatorycomplex, binds to the ends of the 20S core and regulates access ofcellular proteins to the catalytic cavity. The 19S complex, togetherwith the 20S core make up the 26S-proteasome. The 19S complex has atleast 6 distinct ATPase subunits which are thought to promote unfoldingof proteolytic substrates so that they can be channeled through thenarrow pores of the 20S core. The 19S complex contains as many as 20subunits, which include a multiubiquitin-chain binding protein,isopeptidases and at least 6 ATPases. To date, many of these additionalsubunits remain uncharacterized.

[0010] The Rad23 gene of S. cerevisiae is necessary for efficientnucleotide excision repair of damaged DNA. In vitro studies indicatethat this factor may play a role in assisting the assembly of the repaircomplex at the site of damage. Accordingly, interactions between Rad23pand other repair proteins including Rad4p, Rad14p, and subunits of TFIIHhave been proposed. Thus far, however, the exact biochemical function ofRad23p in DNA repair has remained unclear.

[0011] Rad23p has an NH₂-terminal domain with striking homology toubiquitin (22% identity, 43% homology). Watkins et al. have shown thatthis ubiquitin-like domain is required for repair activity of theprotein and that the domain can be replaced by the sequence of wild-typeubiquitin. In addition, a family of proteins with similar ubiquitin-likedomains have been discovered. Unfortunately, these family members havediverse species of origin and apparently disparate functions and thushave provided no clue as the exact role of this domain.

[0012] As noted above, impaired activity of the proteasome is implicatedin many diseases in humans. This observation has stimulated considerableresearch activity in the identification of novel therapeutic agents forinhibiting and/or stimulating the activity of the proteasome. Thesestudies have been hindered by the inefficient, time-consuming,biochemical protocols available for the purification of proteasomes. Thepresent invention describes a rapid and efficient proteasomepurification method and provides novel methods of use of variousproteasome subunits so purified.

SUMMARY OF THE INVENTION

[0013] The present invention provides compositions and a rapid andefficient method for the purification of proteasome complexes from avariety of cell types. In accordance with the present invention, it hasbeen discovered that the ubiquitin-like N-terminal domain of a yeastprotein, Rad23, has high affinity for the proteasome. Accordingly, thisdomain or homologues thereof may be immobilized to a suitable solidsupport and used to isolate the proteasome from cell lysates. Followingremoval of non-specifically bound proteins, the proteasomes are eluted.This method will facilitate the molecular characterization of the as yetunidentified subunits of the proteasome. Ubiquitin-like domains (UbL) incellular proteins vary slightly between species. In one embodiment ofthe invention, UbL-domains from a given species will be used forproteasome purification from cell lysates derived from cells of thatspecies.

[0014] Another aspect of the invention is a kit of materials useful inperforming the proteasome purification method of the invention. A kitaccording to this aspect of the invention comprises a solid support towhich a UbL of interest has been affixed as well as suitable buffers foreluting proteasome preparations.

[0015] In a further embodiment of the invention, it has been discoveredthat this same N-terminal ubiquitin like domain of Rad23, UbL^(R23),functions as a degradation signal in actively growing cells. Fusionproteins comprising this domain are provided herein. Reporter proteinsattached to the UbL domain (UbL^(R23)-reporter) are rapidly degraded inlogarithmically growing cells. Since a primary feature of malignantcells is the aberrant rate of cell growth, the UbL^(R23)-reporterprovides a powerful way to assess the proliferative potential of tumorcells. In yet another embodiment of the invention, the efficacy ofanti-cancer drugs can be assessed by determining the stability of theUbL^(R23)-reporter fusion proteins.

[0016] In a further aspect of the invention, compositions and methodsare provided for enhancing the thermostability of fusion proteinscontaining the UbL domain. Such fusion proteins may be used to advantagein chemical reactions requiring thermostable reagents, such as thepolymerase chain reaction (PCR). In this embodiment of the invention,DNA constructs are generated wherein a DNA sequence encoding aUbL-domain is operably linked to a DNA sequence encoding the protein tobe thermostabilized using standard molecular biological techniques.Following expression of the DNA construct in a suitable host cell, thethermostable fusion protein is purified and utilized in biochemicalassays requiring high temperatures.

[0017] In summary, the methods and kits of the invention areparticularly useful for the assessing proteolytic degradation ofcellular components via the proteasome. The DNA constructs of theinvention encoding fusion proteins comprising UbL domains are useful forassessing the proliferative potential of malignant cells. UbL domainsmay also be utilized to enhance the thermostability of fusion proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1A-1E are an autoradiograph and western blots of cellextracts showing that Rad4-HA interacts with Rad23 and that Rad23interacts with the 26S proteasome. Rad4 plays a role in DNA repair andstably interacts with Rad23. FIG. 1A depicts the positions of [³⁵S] GSTfusion proteins and [³⁵S] Rad-HA. Yeast strains simultaneouslyexpressing the Rad4-HA and each of the GST-fusion proteins weremetabolically labeled with ³⁵S-methionine for 10 minutes. Extracts wereprepared and adsorbed to glutathione-Sepharose. Beads were washedextensively and bound proteins resolved by SDS-PAGE and detected byfluorography. FIG. 1A, 1B and 1C, Lane 1, GST; lane 2; GST-Rad23; lane 3GST^(AUbL)RAd23 and lane 4, GST-UbL^(R23). Rad4-HA is detected in lanes2 and 3 indicating that it interacts with C-terminal sequences in Rad23.Non-specific interactions of other cellular proteins with GST-UbL^(R23)are indicated by asterisks. FIG. 1B is an immunoblot showing that Cim5and Cim3 interact with GST-Rad23 and GST-UbL^(R23) FIG. 1C shows thatthe complex that interacts with GST-Rad23 and GST-UbL^(R23) contains the20S subunit Pup1-HA. The additional band (asterisk) may represent aprecursor form of Pup1-HA. FIG. 1D is a blot showing that native Rad232can be precipitated on FLAG-agarose beads in extracts derived from ayeast strain expressing Prel-FLAG, an epitope tagged derivative of a 20Sβ-subunit. FIGS. 1D and 1E, lane 1, extract from Pre1-flag cells; lane 2extracts from negative control extract lacking Pre-1-FLAG. FIG. 1E is ablot showing that extracts containing FLAG-Rad23 can specificallyprecipitate Cim3 and Cim5 on FLAG-agarose beads. These subunits were notrecovered from extracts containing a control vector lacking FLAG-Rad23(lane 2).

[0019]FIG. 2 is a series of graphs depicting the proteolytic activityassociated with UbL^(R23). Flag-Rad23 was immunopurified and incubatedwith peptide substrates. A control reaction with a strain expressing anunrelated protein is also shown (Negative). The relative levels ofchymotrypsin, trypsin and PGPH-like activities, and the effect ofproteasome inhibitors MG132 and lactacystin are shown. A (−) symbolindicates the absence of the inhibitor. The values represent the averageof three measurements.

[0020]FIG. 3 is fractionation data showing that Gst-R23, Rad4-HA andCim5 are components of a high molecular weight complex. FIG. 3A is aCoomassie stained gel showing Mono-S fractions isolated followingincubation with glutathione-sepharose. Bound proteins were separated bySDS-PAGE. FIGS. 3A, 3B, and 3C show western blots treated sequentiallywith antibodies against HA (FIG. 3A), Cim5 (FIG. 3B) and Rad23 (FIG.3C). FIG. 3D is a graph showing the ATPase activity corresponding to theMono-S fractions isolated.

[0021]FIG. 4 is a blot showing that human HHR23-B interacts with Mss1.The ubiquitin-like domain of HHR23-B was linked to Gst (Gst-Ub^(HRB))and incubated with Hela cell nuclear extracts. Mss1 was detected withCim5 antibodies (lane 2), Cim5 interaction with Gst-R23 is also shown(lane 3).

[0022]FIGS. 5A and 5B are data showing the genetic interaction betweenRAD23 and the N-end rule pathway. FIG. 5A shows that toxicity in yeastis caused by overexpressing the N-end rule pathway (CSY13, top). Thistoxicity is suppressed by high levels of RAD23 (CSY41, left), or inrad23Δ (CSY41, right). Isogenic yeast strains were grown in minimalmedium containing galactose and lacking appropriate nutrients tomaintain plasmids. FIG. 5B is a graph showing that Rad23-ha cancomplement rad23Δ. Exponential-phase yeast cells (JD47-13C; RAD23,closed squares, CSY85; rad23Δ, closed circles, and CSY131; CSY85expressing Rad23-ha, open circles) were exposed to 15, 45 and goJ/m² UVlight (n=3).

[0023]FIG. 6 is an autoradiograph showing the results of pulse-labelingexperiments which indicate that Rad23-HA is not degraded by Ubc4 or theN-end rule system. FIG. 6A shows that Rad23-HA is unstable in ubc4Δubc5Δ suggesting that these E2 proteins do not affect its stability.Rad23-ha is also unstable in N-end rule pathway mutants, ubc2Δ, FIG. 6Band ubr11Δ, shown in FIG. 6C. The stability of Rad23-HA was comparableto that observed in the parental strain (FIG. 7A).

[0024] FIGS. 7A-7D are gels showing the growth-stage specificdegradation of Rad23-HA. FIG. 7A: Rad23-HA stability was measured inlogarithmic- and stationary-phases of growth. The numbers at the topindicate minutes in chase medium. Rad23-HA (arrow) and asepharose-interacting yeast protein (*) are indicated in this andsubsequent figures. FIG. 7B: Stationary-phase yeast cells were labeledfor 20 minutes and extracts were prepared to monitor the abundance of³⁵S-Rad23-HA. The numbers at the top refer to samples withdrawn duringthe labeling (in minutes), and those indicated as + refer to minutes inchase medium lacking ³⁵S-label. Total ³⁵S-protein was also resolved on asecond gel to follow the levels of other cellular proteins (data notshown). FIG. 7C: The stability of Rad23¹⁻³⁶⁹ is shown in logarithmic-and stationary-phase cells. This C-terminal truncated allele does notpossess a HA epitope but displays growth-stage specific degradationsimilar to Rad23-HA (FIG. 6A above) and UbL^(R23)-LacZ (FIG. 9B). FIG.7D shows the degradation of other substrates of the ubiquitin system areunaffected by growth conditions. (R-β-gal is a substrate of the N-endrule pathway while ubiquitin-proline-β-gal is a substrate for theubiquitin-fusion degradation (UFD) pathway. Met-β-gal is not a substrateof either pathway and therefore is stable in both logarithmic andstationary phase cells.

[0025] FIGS. 8A-8C are gels showing that transient growth-arrest doesnot affect Rad23-HA stability. The growth of exponential stage cells wasarrested and Rad23-HA stability was measured. FIG. 8A: RYB 262 containsa temperature-sensitive allele of RNA polymerase II. The growth of RY262expressing Rad23-HA was arrested at 37° C. and pulse-chase analysis wasperformed. FIG. 8B: Hydroxyurea was added to exponentially growing cells(JD47-13C) expressing Rad23-HL and incubated for 2 hours at 30° C.Pulse-chase analysis was carried out when approximately 75% of the cellshad arrested growth. FIG. 8C: A bar1-1 strain expressing Rad23-HA wasexposed to 10 ng/ml α-factor, and pulse-chase analysis was performedwhen approximately 95% of the cells had arrested in G₁.

[0026]FIGS. 9A and 9B are gels showing that UbL^(R23) is a regulated andportable degradation signal. FIG. 9A: ^(ΔUbL)Rad₂₃-HA was expressed inJD47-13C and stability was compared to Rad23-HA in exponential phase.FIG. 9B: UbL^(R23) was linked to β-galactosidase and the stability ofUbL^(R23)-LacZ was determined in JD47-13C. A cluster of protein bandscorresponding to UbL^(R23)-LacZ was detected in stationary-phaseextracts and are indicated by the bracket. UbL^(R23)-LacZ was almostundetectable in exponential-stage cells.

[0027] FIGS. 10A-10E show that Ufd5 is required for the degradation ofRad23-HA. Rad23-HA was expressed in a set of strains bearing mutationsin ufd1-5. In vivo stability was measured by pulse-chase methods andquantitated by PhosphorImager. Only ufd5Δ was found to be important forRad23-HA degradation. An antibody cross-reacting band (*) served as auseful internal control for loading.

[0028] FIGS. 11A-11E are a series of gels showing that Rad23-HA isdegraded by the proteasome. The in vivo stability of Rad23-HA inproteasome and vacuolar mutant strains is shown. FIG. 11A,pre1-1/pre2-2; FIG. 11B, Cim5; FIG. 11C, doaΔ-1; FIG. 11D. mcb1Δ; FIG.11E, pep4Δprb1Δ. An arrow indicates the position of Rad23-HA. A proteinof approximately 70 kD which binds Sepharose non-specifically isindicated by the asterisks.

[0029]FIGS. 12A and 12B are a sequence alignment and graph showing thatRad23 interacts with a putative subunit of the 26S proteasome. Rad23pwas linked to lexA and Irt1 was isolated in a 2-hybrid experiment. FIG.12A shows the amino acid sequence corresponding to the ATPase domain ofIrt1 is aligned with the sequence of closely related homologs of 26Sproteasome subunits. FIG. 12B is a graph showing that the interactionbetween Rad23 and Irt1, and 3 C-terminal truncated alleles of Irt1, asdetermined by measuring β-galactosidase activity in the 2-hybrid yeaststrain harboring both plasmids. The data are representative of 6independent measurements and are indicated in Miller units.

[0030]FIGS. 13A and 13B are gels showing that UbL's from differentsources interact with the proteasome. GST linked proteins were expressedin yeast and purified on glutathione-Sepharose. Proteins retained on thebeads were resolved in SDS-polyacrylamide gels, transferred tonitrocellulose, and incubated with antisera specific to proteasomesubunits Cim3 and Cim5. The blot was developed by enhancedchemiluminescence (Amersham). FIG. 13A: Lane 1 contains a GST control,and lanes 2-6 contain GST linked to UbL^(R23), Ub, UbL^(DSK), UbL^(HRA)and UbL^(HRB). Yeast strains expressing the GST linked proteins as wellas Pre1-Flag, FIG. 13B. The blot was developed with anti-Flag antiserum(Kodak). The position of molecular weight markers are indicated.

[0031]FIGS. 14A and 14B are a series of blots showing that UbL'sinteract with the 19S/PA700 complex. In FIG. 14A GST-UbL^(HRA) andGST-UbL^(HRB) were purifed and incubated with Hela cell S100 extract.Lane 1 contains a GST negative control, while lanes 2 and 3 containGST-UbLHRA and -UbL^(HRB). Lane 4 contains GST-UbL^(R23) interactingproteins. Hela S100 extracts were incubated with GST, GST-UbL^(HRA) andGST-UbL^(HRB) and bound proteins were separated by SDS-PAGE and thewestern blot incubated with Cim5-specific antibodies, which crossreactswith the human counterpart Mss1. In FIG. 13B, a similar set of GSTlinked proteins were incubated with purified 19S/PA700 and the boundproteins separated by SDS-PAGE and visualized by staining with silvernitrate. The profile of subunits that comprise the 19S/PA700 particle isshown in lane 1. Molecular weight standards are indicated in lane 5.(Lanes 1-5 were from the same gel).

[0032]FIG. 15 is a gel showing that UbL^(R23) can target heterologousproteins to the proteasome. UbL^(R23) was linked to β-galactosidase andUra3-HA and transformed into yeast cells expressing GST-Cim5. Lane 1contains affinity purified GST-Cim5. UbL^(R23)-βgal was immunopurifiedwith anti-βgalactosidase antibodies and GST-Cim5 was co-purified (lane2). An extract containing only GST-Cim5 was treated with anti-62galactosidase antibodies (lane 3). UbL^(R23)-Ura3-HA was precipitatedwith anti-HA antibodies and GST-Cim5 was co-purified (lane 4). Anextract containing only GST-Cim5 was incubated with anti-HA antibodiesand resolved as a negative control (lane 5). The blot was developed withanti-GST antibodies. (H and L indicate the positions of immunoglobulinheavy- and light-chains from the HA immunoprecipitation).

[0033]FIGS. 16A and 16B are a pair of gels illustrating that UbL^(R23)interferes with the degradation of specific substrates. Yeast cellsexpressing a test protein Met-βgal, or substrates of the N-end rule(Arg-βgal and Leu-βgal) and UFD pathway (Ub-Pro-βgal) were transformedwith plasmids expressing GST or GST-UbL^(R23). The stability of theproteins was determined by ³⁵S-pulse-chase methods. Samples wereanalyzed after 0, 10 and 60 min in Chase medium containingcycloheximide. The precipitated proteins were separated by SDS-PAGE andthe fluorograms exposed to X-ray film. This figure reproduces a darkexposure of the gel to reveal high molecular-weight derivatives ofLeu-βgal and Ub-Pro-βgal (indicated as (Ub)n) in cells expressing GST-UbL^(R23).

[0034]FIG. 17 is a graph showing the CD spectra of Rad23. The data showthe spectra of a typical globular protein. The CD spectra of the proteinis not altered by heating (not shown).

[0035]FIG. 18 is a graph showing the melt profile of Rad23 at 222 nmfrequency. It is significant to note that there is no temperaturedependent unfolding of the protein.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The proteasome is an essential component of the ATP-dependentproteolytic pathway in eukaryotic cells and is responsible for thedegradation of most cellular proteins. The 20S (700 kDa) proteasomecontains multiple peptidase activities that function through a new typeof proteolytic mechanism involving a threonine active site. The 26S(2000 kDa) complex, which degrades ubiquitinated proteins, contains, inaddition to the 20S proteasome, a 19S regulatory complex composed ofmultiple ATPases and components necessary for binding proteinsubstrates. The proteasome has been highly conserved during eukaryoticevolution, and simpler forms are found in archaebacteria and eubacteria.

[0037] The post-translational attachment of ubiquitin (Ub) to cellularproteins is implicated in a broad range of biological activitiesprimarily involving protein degradation (Hershko 1991). Ubiquitin ismobilized through several trans-thiolation steps which precede itsisopeptide linkage to cellular substrates. Ubiquitin is activated byadenylation of its C-terminal glycine residue by the ubiquitinactivating enzyme, E1 (Hershko 1991). Activated Ub is transferred fromE1 to a family of ubiquitin-conjugating enzymes (E2's or Ubc's) whichplay significant roles in substrate selection. Emerging evidencesuggests that the transfer of Ub to a cellular substrate may require anadditional factor termed E3/Ub-protein ligase (Hershko 1991; Scheffneret al. 1995), or Ubr1/n-recognin (Varshavsky 1992). A well studiedsubstrate targeting mechanism of the ubiquitin system is the N-end rulepathway (Varshavsky 1992), whose overexpression inhibits the growth ofhaploid yeast cells (Madura and Varshavsky, 1994). RAD23, a subunit ofthe nucleotide excision repair complex was isolated in a search forsuppressors of this growth defect. The present invention describes thebiochemical analysis of alleles of Rad23 (Rad23-HA and Rad23-FLAG). Itappears from these studies that Rad23 is involved in both DNA repair andthe ubiquitin protein degradation pathway.

[0038] Previous studies have demonstrated that mutations in RAD23(rad23Δ) result in a defect in the repair of UV-irradiated DNA, which ismanifested by an intermediate sensitivity to DNA damage (Friedberg etal. 1995). The moderate sensitivity of rad23 to UV light is contrastedby the severe defects observed in other excision repair mutants such asrad1, rad2 and rad4 which are unable to incise damaged DNA (Wilcox andPrakash 1981). The removal of DNA lesions is markedly reduced in rad23Δbut not abolished, suggesting that Rad23 plays an accessory role innucleotide excision repair. In vitro studies showed that Rad23 forms astable interaction with the excision repair protein Rad4 (Guzder et al.1995b), although the biological significance of this association isunclear. Rad23 also interacts with other effectors, including the DNAdamage-recognition protein Rad14 and the RNA PolII-specifictranscription factor TFIIH (Guzder et al. 1995a). A previously unknownfunction for Rad23 in spindle-pole body (SPB) duplication was recentlydescribed (Biggins et al. 1996). These results indicate that Rad23 canparticipate in multiple regulatory pathways.

[0039] It has been discovered that the Rad23 N-terminal domain(UbL^(R23)) has a strong affinity for the 26S-proteasome and can be usedto advantage to purify this proteolytic complex in a single step.Immobilizing this domain to a solid support, followed by exposure tocellular lysates results in the retention of the proteasome on thesupport. The proteasome can then be released from the support followingthe prior elution of all other non-specifically adsorbed proteins. Afamily of proteins having Ub-like domains have been observed in avariety of other species from yeast to humans (Toniolo et al. 1988;Wiborg et al., 1985). Ubiquitin-like domains in yeast Rad23 and Dsk2, aswell as human HHR23A and HHR23B, are proteasome-interacting sequences.The attachment of UbL²³ to a reporter protein also targeted it to theproteasome, demonstrating that this is an autonomous function of a UbL.The ubiquitin-like domain of Rad23 (UbL^(R23)) interacts with a complexthat contains subunits of the 26S proteasome and displays ATPase andprotease activities expected for this proteolytic system. In agreementwith this finding, proteasome-specific inhibitors caused markedreduction in the proteolytic activity associated with UbL^(R23). Theubiquitin-like domain of Dsk2 (UbL^(DSK)) binds the proteasomepreferentially in actively growing cells. Overexpression of UbL^(R23)inhibits the degradation of specific substrates of the ubiquitin pathwayperhaps by saturating the proteasome targeting pathway. These resultssuggest that the physiological roles mediated by proteins containingubiquitin-like motifs converge at the level of the proteasome, and mayinvolve distinct proteasome subunits. The use of these ubiquitin-likehomolog sequences for the purification of proteasomes from correspondingcell types, (e.g., human UbL-domains to purify human proteasomes oryeast UbL-domains to purify yeast proteasomes) is contemplated to bewithin the scope of the invention.

[0040] Kits are provided for purifying proteasomes from a variety ofcell types. Such kits would include predetermined UbL domains fused to asolid support. The kit of the invention may also conveniently include adevice for purifying biological samples, together with various solutionswhich may be used in performing the purification procedure, such asbuffer(s), saline, diluent, controls and the like.

[0041] In accordance with another aspect of the present invention, ithas been discovered that the half-life of Rad23-HA is tightly regulated,ranging from approximately 1 minute in actively growing cells to greaterthan 1 hour in stationary-phase. In contrast to the instability of theepitope-tagged Rad23-HA allele, it was previously reported that nativeRad23 is stable (Watkins et al. 1993). Data presented herein reveal thatRad23 is degraded during the G1/S phase of the cell cycle. Specifically,data are described which indicate that the ubiquitin-like domain ofRad23 (UbLR^(R23)) is an autonomous and regulated degradation signal.Two additional lines of evidence suggest a direct interaction with theproteolytic apparatus: 1) Rad23 interacts with Irt1, a protein that hasstrong similarity to ATPase subunits of the 26S proteasome, and 2)immunopurified 26S proteasome contains native Rad23. Although thefraction of Rad23 that is associated with the proteasome is not known,the findings presented herein strongly implicate a proteolytic functionfor Rad23.

[0042] Malignant cells display aberrant growth properties and do notrespond to normal regulatory signals. Malignancy therefore arisesbecause aberrant cells continue to grow in conditions when normal cellsremain quiescent. Detection and treatment of proliferative disordersmust begin with the clear identification of cells that manifest aberrantgrowth rates. Although malignant cells are often morphologicallydistinguishable from their wildtype counterparts, a quantitativemeasurement of the growth properties of cells is lacking.

[0043] In another embodiment of the present invention, methods areprovided which employ UbL^(R23)-LacZ fusion protein(s) to assess cellgrowth rates in evolutionarily divergent organisms from yeast to humans.UbL-fusion proteins in rapidly dividing cells are degraded rapidlywhereas those in quiescent cells remain stable. Proliferative rates arethen determined based upon the half life of the fusion protein withinthe cell. Additionally, UbL^(R23) can be linked to selectable markers,as well as genes that confer drug resistance. In these types of assays,cells that stabilize a fusion protein produced from a DNA constructcomposed of UbL^(R23) coding sequence linked to a drug resistance genewould survive in the presence of the drug. It is expected thatproliferating cells will actively degrade the fusion protein and succumbto the presence of the drug. In a quantitative assay such as this, dosetitrations are employed to define the conditions that promote thekilling of malignant cells without harming normal cells.

[0044] The ubiquitin-like domain UbL^(R23) has been operably linked tothe reporter protein β-galactosidase (UbL^(R23)-LacZ) to demonstrate thefeasibility of this concept. In earlier studies performed in thislaboratory, growth dependent degradation of native Rad23 was observed.These data suggested that UbL^(R23) was an important component of thedegradation signal. As proposed, UbL^(R23)-LacZ fusion proteins provedto be exceedingly unstable in actively growing cells but entirely stablein quiescent cells, mimicking the degradation profile of Rad23 protein.

[0045] To further assess the suitablity of using UbL^(R23)-LacZ fusionproteins to assess growth potential, this fusion protein was produced incells expressing various Ras mutants. Ras proteins are highly conservedsmall GTP-binding regulators that control growth, differentiation and avariety of other cellular functions. Oncogenic alleles of Ras arehyperactive and do not arrest growth properly, while null mutants of Rasarrest growth prematurely. The data revealed that the level ofUbL^(R23)-LacZ was almost undetectable in a strain expressing theoncogenic Ras mutant, while elevated levels of UbL^(R23)-LacZ weredetected in cells lacking Ras. These findings corroborate the proposalthat UbL^(R23)-LacZ is a suitable reporter protein to assess theproliferative potential of cells.

[0046] The strategy described above enables the identification ofgenetic mutants that promote or attenuate the degradation of theUbL^(R23)-linked chimeras. It is anticipated that such mutants wouldeither promote or inhibit proliferation. This method also provides a wayto screen for compounds that promote quiescence. For instance, ifUbL^(R23) is linked to a gene that confers drug resistance, theexpression of drug resistance should be confined to quiescent cells, orcells whose growth has been artificially arrested.

[0047] In yet another aspect of the present invention, it has beendiscovered that the UbL^(R23) domain confers thermostability on Rad23and on fusion proteins to which this domain has been operably-linked.Thus the UbL domain is a cis-acting temperature stabilizer. This domaincan be used to advantage to create fusion proteins with enhancedthermostability.

[0048] PCR assays utilize the Taq polymerase enzyme which functions atthe higher temperatures required for PCR yet also generates errors inthe amplified sequences as the enzyme exhibits reduced fidelity in DNAcopying. In one embodiment of the invention, the UbL domain may be fusedto a polymerase enzyme which has a reduced error rate. Such fusionproteins can be used in PCR assays to increase the fidelity of DNAamplification.

[0049] The definitions set forth below are provided to facilitateunderstanding of the subject matter of the present invention:

[0050] The term proteasome refers to a 26S multicatalytic protease.

[0051] The phrase N-end rule pathway relates the in vivo half-life of aprotein to the identity of its amino-terminal residue. Overexpression oftargeting components of the N-end rule pathway in S. cerevisiae inhibitsthe growth of yeast cells.

[0052] The term promoter region refers to the 5′ regulatory regions of agene. In the present invention, the use of both strong constitutive genepromoters and inducible gene promoters is contemplated.

[0053] The term operably linked means that the regulatory sequencesnecessary for expression of the coding sequence are placed in the DNAmolecule in the appropriate positions relative to the coding sequence soas to effect expression of the coding sequence. This same definition issometimes applied to the arrangement of transcription units and othertranscription control elements (e.g. enhancers) in an expression vector.The term may also be used to describe the fusion of a nucleic acidsequence encoding a UbL domain of the invention to a second nucleic acidsequence encoding a protein of interest. Expression of the fused nucleicacid sequences results in the production of a fusion protein.

[0054] The term fusion protein refers to a chimeric protein moleculecomprising two or more domains from different sources.

[0055] The term DNA construct refers to genetic sequence used totransform cells. These constructs may be administered to cells in aviral or plasmid vector.

[0056] The term reporter gene refers to a gene whose expression may beassayed; such genes include, without limitation, lacZ, amino acidbiosynthetic genes, e.g., the yeast LEU2, HIS3, LYS2, or URA3 genes,nucleic acid biosynthetic genes, the mammalian chloramphenicoltransacetylase (CAT) gene, the green fluorescent protein (GFP) or anysurface antigen gene for which specific antibodies are available.

[0057] The term selectable marker gene refers to a gene product thatwhen expressed confers a selectable phenotype such as antibioticresistance on a transformed cell.

[0058] Methods of delivery of the DNA constructs of the invention totarget cells include electroporation, CaPO₄ precipitation, lipid-basedsystems and microinjection. Standard methods for delivery of DNA andprotocols for preparing the transforming DNA may be found in CurrentProtocols in Molecular Biology, eds. Frederick M. Ausubel et al., JohnWiley & Sons, 1995.

[0059] The following specific examples are provided to illustratevarious embodiments of the invention. They are not intended to limit thescope of the invention in any way.

EXAMPLE I Rapid and Efficient Purification of Proteasomes Using Rad23and Components Thereof

[0060] Rad23 has an unusual N-terminal domain that bears a strikingresemblance to ubiquitin (Watkins et al., 1993). This domain, which hasbeen designated UbL^(R23), is important for DNA repair because itselimination causes sensitivity to UV light (Watkins et al., 1993). Arole for Rad23 in the ubiquitin system was suggested by its suppressionof N-end rule induced toxicity, which raised the possibility of aproteolytic function in DNA repair.

[0061] Rad23 and Rad4, a well as the human counterparts HHR23-B and XPC,form stable interactions. We therefore tested whether GST-Rad23interacts with components of the DNA repair and proteolytic pathways, Welinked Rad4 to the HA epitope (SEQ ID NO:17:Try-Pro-Try-Asp-VAl-Pro-Asp-Tyr-Ala(Rad4-HA) and found that itcomplemented rad4Δ. GST-Rad23 and RAd4-HA were expressed simultaneouslyin yeast cells and metabolically labeled with [³⁵S] -methionine.Radiolabeled extracts were applied to glutathione-Sepharose and boundproteins analyzed by SDS-PAGE and fluorography. Rad4-HA interacts withGST-R23. See FIG. 1A, lane 2. The interaction of Rad4-HA with GST-Rad23did not require UbL^(R23), (FIG. 1A, lane 3) demonstrating that distinctregions of Rad23 interact with the proteolytic and DNA repair pathways.Identical samples were transferred to nitrocellulose and analysed byincubation with anti-HA antibodies and, consistent with these findings,RAd4-HA was detected only in lanes 2 and 3. These findings are inagreement with a recent report showing that 21 C-terminal residues inRad23 are important for interaction with Rad4.

[0062] To further explore the proteolytic function of Rad23 in DNArepair, Rad23 and two truncated mutants were operably linked toglutathione-S-transferase (GST-Rad23, GST-^(ΔUbL)Rad23, andGST-UbL^(R23)), and immobilized on glutathione-Sepharose. Western blotscontaining the proteins released from GST and GST-Rad23 beads wereincubated with antibodies against Cim3 (Sug1) and Cim5. Cim3 and Cim5are ATPases of the regulatory (19S) subunit of the 26S proteasome. BothCim3 (M_(r) 43K) and Cim5 (M_(r) 54K) were detected in the GST-Rad23beads (FIG. 1B, lane 2) but not in the control GST beads (FIG. 1B, lane1). GST-UbL^(R23) alone could efficiently bind a complex containing Cim3and Cim5 (FIG. 1B, lane 4) but, a mutant lacking UbL^(R23)(GST-^(ΔUbL)Rad23) could not (FIG. 1B, lane 3). Two variants of Rad23,bearing small epitopes on either the N-terminus (FLAG-Rad23, FIG. 1e) orthe C-terminus (Rad23-HA, data not shown) also interacted with theproteasome. Both Cim3 and Cim5 were detected in anti-FLAGimmunoprecipitates prepared from yeast cells expressing FLAG-Rad23 (FIG.1E). Because yeast cells expressing ^(ΔUbL)Rad23 fail to complementrad23Δ, these findings suggest that Rad23-proteasome interaction isimportant for DNA repair. These data also show that UbL^(R23) representsa new proteasome interaction signal. A large family of proteins bearingubiquitin-like extensions have been identified, and our results suggestthat they too have proteolytic functions.

[0063] To determine whether the GST-Rad23 interacting complex included20S catalytic subunits, extracts from cells expressing Pre1-FLAG (28K)or Pup1-HA (33K), both of which are epitope-tagged derivatives of 20Sβ-subunits were analyzed. Both Pup1-HA (FIG. 1C, lane 2) and Pre1-Flag(data not shown) were detected in GST-Rad23 beads after incubation withFLAG or HA antibodies, confirming the presence of 20S catalyticsubunits. GST-UbL^(R23) accumulated to higher levels than thanGST-Rad23, and the recovery of Pup1-HA was proportionately higher (FIG.1C, compare lanes 2 and 4). To confirm that the findings applied tonative Rad23, Pre1-FLAG was immunoprecipitated on FLAG-agarose beads andinteracting proteins were resolved on SDS-PAGE. Proteins weretransferred to nitrocellulose and the blots were incubated withRad23-specific antibodies. Native Rad23 was readily detectable inimmunoprecipitates containing Pre1-FLAG but not from a control extractlacking this epitope-tagged proteasome subunit (FIG. 1D). Approximately5% of cellular Rad23 precipitated with Pre1-FLAG. This estimate is basedon the amount of Rad23 that remained on the FLAG-agarose beads after 18hours at 4° C. The in vivo interaction could be higher if theinteraction with the proteasome is transient or regulated.

[0064] To examine if the Rad23 interacting complex hadproteasome-specific activities we measured ATPase (Merrick, W. C.,1979), and protease activities (Heinemeyer et al., 1991). We found thathigh levels of ATPase activity were associated with FLAG-Rad23 (Kibel etal., 1995). Consistent with this finding, high proteolytic activity wasdetected against three different peptide substrates in FLAG-Rad23immunoprecipitates. This activity was significantly reduced by theproteasome inhibitors MG132 and lactacystin (Coux et al., 1996). SeeFIG. 2.

[0065] To characterize the interaction of Rad4 with Rad23, extracts wereprepared from cells expressing both GST-Rad23 and Rad4-HA and proteinsseparated on Sephacryl S-200. GST-Rad23 was detected in the void volumecoincident with dextran blue, and also in fractions corresponding to itspredicted monomeric size (approximately 80K). GST-Rad23, Cim5 andRad4-HA could each detected in the high molecular weight fraction,suggesting that they are components of a single complex. See FIGS. 3A-C,lane 2.

[0066] To investigate this further, proteins in the Sephacryl S-200 voidvolume were chromatographed on Mono-Q. GST-Rad23, Rad4-HA, and Cim5 weredetected in samples eluting at approximately 0.35M KCl. Significantly,these fractions were previously shown to contain catalytically activeproteasome (Rubin et al., 1996). Fractions that eluted between 325 and375 mM KCl from the Mono-Q column were pooled and chromatographed onMono-S. Cim5 and Rad4-HA again co-fractionated with with GST-Rad23(FIGS. 3A-3C, lanes 15-18) and a peak of ATPase activity copurified withthe GST-Rad23 interacting complex. See FIG. 3D.

[0067] As mentioned previously, two human homologues of Rad23, HHR23-Aand HHR23-B contain N-terminal ubiquitin-like domains, suggesting thatthey act in a similar way to the yeast protein, Rad23. SignificantlyHHR23-B forms a stable interaction with XPC, the human counterpart ofRad4. To explore the functional relatedness among this class ofproteins, the ubiquitin-like domain of HHR23-B (UbL^(HRB)) was linked toGST. GST-UbL^(HRB) was immobilized on glutathione sepharose and reactedwith nuclear extracts prepared from Hela cells (a gift from D. Reinberg,RWJMS, New Jersey). Cim5 antibodies revealed an interaction betweenGst-UbL^(HRB) and Mss1, the human equivalent of Cim5. See FIG. 4, lane2. GST-Rad23 interaction with Cim5 (lane 4) confirmed the specificity ofthe antibody reaction. The evolutionary conservation of yeast and humanDNA repair and ubiquitin pathways strongly suggests that the molecularinteractions reported here are evidence of a novel mechanism forregulating DNA repair in yeast and humans. These findings also indicatethat ubiquitin-like sequences represent a novel class ofproteasome-interacting domains, and their characterization mayfacilitate the molecular elucidation of the mechanistic action ofproteins that bear this domain.

[0068] As exemplified herein, UbL-like domains can be used toefficiently purify the proteasome. This rapid purification methodenables purification from a variety of cell types. The UbL-domains maybe immobilized to a solid support such as an immunoaffinity column.Following immobilization, the column is exposed to cell lysates,non-specific proteins are eluted and the immobilized proteasomesubsequently purified.

[0069] Exemplary UbL-domain containing sequences for use in the methodsof the present invention are set forth below: DB M Q     I F V K T L T GK T I T L E V E P S D T I E N V K A K I Q D K E G I   P P DSK M S L N IH I K S G Q D K W E V N   V A P E S T V L Q F K E A I N K A N G I   P VRAD M V S L Y F   K N F K K E K V P L D L E   P S N T I L E T K T K L AQ S I S C E E HRB M Q V     T L K T L Q Q Q T F K I D I D P E E T V K AL K E K I E S E K G K   D A HRA M A V T I T L K T L Q Q Q T F K I R M EP D E T V K V L K E K I E A E K G R   D A 212 A V H L T L K K I Q A P KF S I E H D F S P S D T I L Q I K Q H L     I S   E E K A RUB1 M I     VK V K T L T G K E I S V E L K E S D L V Y H I K E       L L       E E KE 173 E E I A A F   R I F R K K S T S N L K S S H T T S N L V K K T M FK R D L L K Q D DE D         Q Q R L I F A G K Q L E D G R T L S D Y N IQ K E S T   L E L V L R L R G G DSK A N         Q R L I Y S G K I L K DD Q T V E S Y E I Q D G H S   V H L V K S Q P K P RAD S         Q I K LI Y S G K V L Q D S K T V S E C G L K D G D Q V V F M V   S Q K K S HRBF P V A G   Q K L I Y A G K I L N D D T A L K E Y K I D E K N T V V V MV T K P K A HRA F P V A G   Q K L I Y A G K I L S D D V P I R D Y R I DE K N F V V V M V T K T K A 212 S H I S   E I K L L L K G K V L H D N LF L S D L K V T P A N S T I T V M I K P N P T S ROB1 G I P P S Q Q R L IF Q G K H S D D K L T V T D A B L V K G M Q L K L V L T L R G G 173 P KR K L Q L Q Q R F A S P T D R L V S P C S L K L N E H K V K M F G K K KK V N P M

[0070] Sequences listed above: SEQ ID NO: 1 Ub: ubiquitin SEQ ID NO: 2DSK: yeast Dsk2 SEQ ID NO: 3 RAD: yeast Rad23 SEQ ID NO: 4 HRB: humanRad23-B (HHR23-B) SEQ ID NO: 5 HRA: human Rad23-A (HHR23-A) SEQ ID NO: 6212: yeast protein of unknown function that contains an internal UbL SEQID NO: 7 RUB1: yeast ubiquitin-like protein that is post-translationallyconjugated to other proteins SEQ ID NO: 8 173: yeast protein of unknownfunction that contains an internal UbL

[0071] Additional ubiquitin-like domain sequences for use in the methodsof the present invention are set forth below: SUMO1MSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFK SEQ ID NO:9VKMTTHLKKLKESYCQRQGVPMNSLRFLFEGQRIADN HTPKELGMEEEDVIEVYQEQTGGHSTV SMT3BMADEKPKEGVKTENNDHINLKVAGQDGSVVQFKIKRH SEQ ID NO:10TPLSKLMKAYCERQGLSMRQIRFRFDGQPINETDTPA QLEMEDEDTIDVFQQQTGGVY SMT3AMSEEKPKEGVKTENDHINLKVAGQDGSVVQFKIKRHT SEQ ID NO:11SLSKLMKAYCERQGLSMRQIRFRFDGQPINETDTPAQ LRMEDEDTIDVFQQQTGGVPE SMT3 yeastMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFF SEQ ID NO:12KIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQA DQTPEDLDMEDNDIIEAHREQIGGAT

[0072] SMT3: yeast ubiquitin-like protein that is post-translationallyconjugated to other proteins like ubiquitin and RUB1. SUMO: mammalianhomolog of the yeast SMT3 Elongin: mammalian protein containing UbL thatis not conjugated to other proteins parkin: UbL-containing proteinimplicated in juvenile Parkinson's disease

EXAMPLE II Rad23 and Its Role in Protein Degradation

[0073] Nucleotide excision repair is enhanced by Rad23, a member of aclass of proteins that bear unusual ubiquitin-like extensions at theirN-termini. Specific modifications of Rad23 cause rapid degradation viathe ubiquitin/proteasome system. Surprisingly, the short in vivohalf-life of these variants does not affect the DNA damage response andcan be reconciled with a growth-stage specific function for Rad23. Thedegradation signal in Rad23 resides in its N-terminal ubiquitin-likedomain (UbL^(R23)), which confers instability when placed on aheterologous protein. Evidence for a proteolytic function for Rad23 issuggested by its interaction with Irt1p, a protein that bears a strikingresemblance to members of the ATPase subunits of the 26S proteasome.Rad23 can be co-precipitated with immunopurified 26S proteasome,implicating a proteolytic function during DNA repair.

Materials and Methods for Example II Isolation of High-copy Suppressorsof N-end Rule Overexpression

[0074] Yeast strain KMY950 was generated by transforming JD47-13C with a2 μm-based plasmid expressing UBR1 and UBC2 from the galactose-inducibleGAL1/10 promoter. The growth of KMY950 is severely impaired ongalactose-containing medium due to overexpression of the N-end rulepathway (Madura and Varshavsky 1994). KMY950 was transformed (Gietz etal. 1992) with a plasmid library expressing yeast cDNAs from the GAL1promoter (Liu et al. 1992). Based on control plating experiments, weestimated that a total of approximately 10⁵ transformants were analyzed.Plasmid DNAs that enabled KMY950 to grow on galactose-containing mediumwere identified and subjected to sequence analysis by the dideoxychain-terminating method. One strong suppressor (plasmid pCEP10) encodedthe complete open reading frame of the yeast RAD23 gene.

Strains, Media, Growth Conditions and Genetic Techniques

[0075]S. cerevisiae strains include JD47-13C (MATa his3-Δ200 leu2-2, 112ura3-52 trp1-Δ63 lys2-801); CSY85 (rad23Δ::URA3 in JD47-13C); CSY228(5-FOA cured ura⁻ derivative of CSY85); BR4 (MATΔ pre1-1 pre2-2 ura3-Δ5leu2-2, 112 his3-11, 15); RY262 (MATΔ his4-518 ura3-52 rpb1-1); BJ5457(MATα ura3-52 trp1 lys2-801 leu2-Δ1 his3-Δ200 pep4::HIS3 prb1Δ1.6Rcan1); Y791 (MATa his3-Δ200 leu2-Δ1 ura3-2 cim5-1); KMY334 (MATa his7cdc7-4 ura3 bar1-1); CTY10-5d (MATa ade2 gal4 gal80 his3-Δ200 leu2-3,112 trp1-Δ901 URA3-lexop GAL1-LacZ). The ubc4Δ, ubc5Δ, ubc4Δ ubc5Δ andthe congenic wildtype strains have been described previously (Chen etal. 1993). A rad4Δ::URA3 deletion was made in MKP°; (MATα ade2 lys2can1-100 his3-Δ200 ura3-52 trp1-Δ901 leu2-2, 112). E. coli strainMC1066, bearing the pyrF74:pn5 mutation was used to select plasmidsexpressing yeast URA3. Yeast growth media were prepared as describedpreviously (Guthrie and Fink, 1991). The expression of genes linked tothe CUP1 promoter was induced by the addition of 0.1 mM CuSO₄. Forpulse-chase analysis exponential-phase cells were grown to a density atA₆₀₀ of approximately 0.5 and stationary phase cultures were grown toA₆₀₀>2.5. In experiments where we measured the stability of Rad23-ha inboth conditions, stationary-phase cultures were collected (25 ml) bycentrifugation, washed and resuspended in a small volume of steriledH₂O. The cell suspension was inoculated into the used stationary-phasemedium and fresh YPD medium, and incubated with vigorous aeration at 30°C. for 4-5 hours to enable the YPD cultures to resume exponentialgrowth. After 4 hours at 30° C. the density of the YPD culture increasedby approximately 2-fold, indicating recovery from stationary phase.

Plasmids, DNA Manipulations and DNA sequencing

[0076] Recombinant methods were performed by standard procedures(Ausubel, 1992). We amplified RAD23 by polymerase chain reaction (PCR)using oligonucleotide primers (#42: 5′-GCGAATTCATGGTTAGCTTAACC-3′ (SEQID NO: 13)and #41: 5′-GCGGTACCCGTCGGCATGATCGCTG-3′) (SEQ ID NO:14). Theprimers introduced an EcoRI site on the 5′ end and a KpnI site on the 3′end of the DNA fragment. A 1.2 kb EcoRI-KpnI PCR DNA fragment wasligated to EcoRI/KpnI-digested pKM1362-2 (Madura and Varshavsky, 1994),yielding plasmid pCS8. In pCS8, Rad23p is linked to a C-terminalHA-epitope (Rad23-ha) and is expressed from the CUP1 promoter. Toconstruct rad23Δ a 4.8 kb EcoRI fragment containing a disrupted alleleof RAD23 was excised from pDG28 (Madura and Prakash, 1990) and used toreplace the wild-type gene in JD47-13C by homologous recombination(Rothstein 1991). The resulting rad23Δ::URA3 strain (CSY85) was platedon 5-FOA containing medium to isolate CSY228, a ura-derivative (Boeke etal. 1984). To make Rad23-ha lacking its N-terminal ubiquitin-like domain(pWP1), DNA sequence encoding codons 78 to 398 were amplified usingoligonucleotide primers (88: 5′ GCGAATTCATGACGAAGACCAAACTAACAGAA-3′; SEQID NO:15, and 41: SEQ ID NO:14) and ligated to pKM1362-2, as describedabove. Similarly, DNA sequence corresponding to codons 1-77 (UbL^(R23))were amplified and ligated to LacZ in pKM1362-2 to yield UbL^(R23)-LacZ.Oligonucleotide primers, specific to the coding sequence ofβ-galactosidase gene (beginning at codon #8), were used to amplify LacZ.

Two-hybrid System Screen and cloning of IRT1

[0077] RAD23 was isolated on a Dra1-EcoR1 DNA fragment, treated with DNAPol1-Klenow, and ligated to similarly treated BamH1 digested pBTM116(Paetkau et al. 1994). The resulting plasmid DNA, encoding lex-Rad23,was transformed into CTY10-5d. Yeast genomic DNA libraries weretransformed into CTY10-5d expressing lexA-Rad23p and approximately2.4×10⁵ transformants were screened to identify blue colonies onindicator plates. Plasmid DNAs were purified from colonies thatdisplayed an interaction (based on the color assay), and were subject toDNA sequence analysis using the primer 5′-GAAGATACCCCACCAAAC-3′, SEQ IDNO:16, and then compared to sequences in GenBank using the BLASTalgorithm. The DNA sequence in plasmid pDG869 corresponded to anopen-reading-frame designated YER047C on Chromosome V. A Lambda cloneencompassing this region (#6379) was obtained from the American TypeCulture Collection, and a 3.2 kb Pst1 DNA fragment was isolated andligated to Pst1 digested pUC19 (pRK1). A 3.5 kb BssS1 DNA fragment waspurified from pRK1, treated with DNA Pol1-Klenow, and ligated to theSma1 site in pUC8 (pRK16). A 3.2 kb EcoR1 DNA fragment was isolated frompRK16 and ligated to EcoR1 treated pGAD424, to generate an in-framefusion of Irt1p to the activation domain of Gal4 (pRK26). To generateC-terminal truncations of Irt1p plasmid pRK26 was treated with Bsu361,Bcl1 and Nde1 and relegated to yield alleles encoding residues 1-567,1-243 and 1-1.72, respectively. Measurement of β-galactosidase activitywere as described in Paetkau et al., 1994.

Pulse-chase and Immunoprecipitation

[0078] Pulse-chase analysis, protein extraction, quantitation andimmunoprecipitation of HA-tagged and β-gal fusion proteins were carriedout as described previously (Madura and Varshavsky, 1994). Yeast cellswere labeled for 5 minutes with ³⁵S-Translabel (ICN Pharmaceuticals),and the reaction was terminated by the addition of buffer containingcycloheximide and excess cold methionine and cysteine.Immunoprecipitations were carried out using equal cpm of lysate (thatwere adjusted to equal volume). Immune complexes were captured onProtein-A Sepharose and resolved on SDS-polyacrylamide gels.Autoradiographic images were quantitated by PhosphorImager analysis ordensitometry. Rad23-HA was detected with HA-specific antibodies(Boehringer Manneheim, Inc.).

UV Irradiation and Survival Measurement

[0079] UV irradiation (at 254nm) and estimation of survival wereperformed as described previously (Wilcox and Prakash 1981). Irradiatedcells were allowed to recover in the dark for 3 days at 30° C.

Cell Cycle Arrest

[0080] PolII^(ts): Rad23-HA was expressed in a strain containing atemperature-sensitive allele of RNA polymerase II (RY262: rpb1-1). RY262expressing Rad23-HA was grown at 23° C. in the presence of 0.1 mM CUSO₄and then diluted 4-fold into YPD (+0.1 mM CUSO₄) that was equilibratedat 37° C., and incubated with vigorous aeration for 2 hours. Cells werecollected by centrifugation and pulse-chase measurements were performedat 37° C.

[0081] Growth arrest with a-factor and hydroxyurea: To measure thestability of Rad23-HA in G₁ arrested cells Rad23-HA was expressed inKMY1012, a ura3 derivative of 4910-3-3A (Madura et al. 1990). KMY1012was grown at 23° C. to A₆₀₀ of approximately 0.3 and then suspended inYPD medium containing 10 ng/ml α-factor (Peninsula Labs). The culturewas maintained at 23° C. for 3 hours until greater than 95% of α-factortreated cells were unbudded and arrested in G₁. Actively growingJD47-13C cells were treated with 100 μg/ml hydroxyurea (Sigma ChemicalCo.) until greater than 75% of the culture displayed large dumbbellshaped cells. The arrested cells were subject to pulse-chase analysis asdescribed earlier.

Rad23 Suppresses N-end Rule Toxicity

[0082] Overexpression of the N-end rule pathway causes growth inhibitionwhich stems, at least in part, from the constitutive degradation of theessential Gα protein (Madura and Varshavsky, 1994). The degradation ofGα is believed to activate the mating-response pathway which causesgrowth arrest in G₁. In a screen to identify high-copy suppressors ofN-end rule dependent toxicity, RAD23 was isolated. See FIG. 5A. Wetheorized that Rad23 might interact with the targeting components of theN-end rule pathway and thereby prevent Gα degradation. Such aninteraction would suggest that Rad23 is a substrate of the N-end rulepathway, or a regulatory component of this proteolytic system. Twocopies of the 9 residue HA epitope were linked to the C-terminus ofRad23. Rad23-HA conferred wildtype levels of UV resistance in rad23Δ,indicating that it is functionally competent. See FIG. 5B.

[0083] The stability of Rad23-HA was measured by pulse-chase analysisand found to be extremely short-lived in wildtype and ubriA cellsindicating that it is not a substrate of the N-end rule pathway,contrary to our prediction. See FIG. 6C. FIG. 6A shows that Rad23-HA isunstable in ubc4Δ ubcΔ suggesting that these E2 proteins do not affectits stability. The degradation of Rad23-ha was also unaffected in ubc2Δ(FIG. 6B), a strain lacking the ubiquitin-conjugating enzyme essentialfor this proteolytic system. The stability of Gα (and other substratesof the ubiquitin pathway), was unaffected in rad23Δ or when Rad23 wasoverexpressed (data not shown), demonstrating that Rad23-mediatedsuppression of N-end rule toxicity does not involve the mating response.Interestingly, it was discovered that rad23Δ also suppressed the toxiceffects of N-end rule overexpression, providing genetic evidence for aconnection between Rad23 and the proteolytic system (FIG. 5A).

Rad23-HA is Conditionally Degraded

[0084] The data demonstrate that the stability of Rad23-HA varied in agrowth-stage dependent manner. The half-life of Rad23-HA exceeded 1 hourin stationary phase cells and was reduced to approximately 1-3 minutesin actively growing cells (FIG. 7A). Even when overexpressed thehalf-life of Rad23-HA was approximately 1 minute during active growth,attesting to the extraordinary specificity and potency of thedegradation apparatus. To exclude the possibility that overall proteindegradation was reduced in stationary-phase cells, thereby causingRad23-HA stabilization, the stability of two distinct classes ofsubstrates of the ubiquitin pathway was examined. Additionally, the invivo half-life of R-βgal and Ub-P-βgal (substrates of the N-end rule andUFD pathways, respectively) were measured. See FIG. 7D. Unlike Rad23-HA,R-βgal and Ub-P-βgal were efficiently degraded in both exponential andstationary-phases of growth. In contrast M-βgal, which is not recognizedas a substrate of the ubiquitin pathway, remained stable in both growthconditions. These results demonstrate that the growth-stage specificdegradation of Rad23-HA (and UbL^(R23)-βgal, described in FIG. 9B) ishighly specific, and is not a reflection of the overall levels ofproteolysis.

[0085] To further characterize the conditions that promote Rad23-HAdegradation, stationary-phase yeast cells were radiolabeled to generatehigh levels of stable Rad23-HA. Rad23-HA was rapidly degraded when thesecells were transferred to rich (YPD) medium, and was undetectable within15 minutes (FIG. 7B). The levels of other proteins were not affecteduntil 60 minutes after transfer (data not shown). The rapid degradationof Rad23-HA precluded our ability to detect multi-ubiquitinatedintermediates.

[0086] The C-terminal HA epitope does not contribute directly to thedestabilization of Rad23-HA because other perturbations of theC-terminus also caused conditional degradation. A Rad23 mutant thatlacked 29 C-terminal residues as well as the HA epitope (Rad23¹⁻³⁶⁹),displayed extreme instability in logarithmic-phase cells (FIG. 7C),resembling the degradation pattern of both Rad23-HA and UbL^(R23)-βgal(FIG. 9B). Significantly, Rad23¹⁻³⁶⁹ conferred UV resistance in rad23Δ(data not shown), suggesting that the function of Rad23 in mediatingprotein degradation is restricted to stationary-phase cells.

Transient Cell-cycle Arrest Does Not Affect Rad23-HA Stability

[0087] The growth-stage dependent degradation of Rad23-HA prompted theexamination of its stability during the cell-cycle. The growth ofexponential stage cultures was arrested with α-factor (Madura andPrakash, 1990) or hydroxyurea (Sanchez et al., 1996), and Rad23-HAstability was determined. Pulse-chase studies revealed that Rad23-HA wasefficiently degraded in these growth arrested cells (FIGS. 8B and 8C).Also a temperature-sensitive allele of RNA Pol II was employed toasynchronously arrest growth of an actively propagating culture (Nonetet al., 1987). The data show that Rad23-HA remained extremelyshort-lived (FIG. 8A). We conclude from these results that thedegradation of Rad23-HA is not affected by transient growth arrest ofexponential-phase cells.

The ubiquitin-like Domain is Required for Rad23-HA Degradation

[0088] Ubiquitin is expressed either as an N-terminal fusion to specificribosomal proteins (Finley et al., 1989), or as a chain oftandemly-linked Ub multimers (Ozkaynak et al., 1990). The C-terminus ofUb is important for its processing, activation and conjugation tocellular proteins. The C-terminal residues in most ubiquitin-likedomains differ from that of Ub suggesting that they are generally notexcised and conjugated to other proteins.

[0089] Varshavsky and colleagues found that the expression of Ub as anon-cleavable extension on β-galactosidase led to extreme instability ofthe fusion protein following subsequent conjugation to a multi-ubiquitinchain (Johnson et al. 1992). Since UbL^(R23) is retained in matureRad23, its role in Rad23-HA degradation was investigated by constructinga mutant that lacked this motif (^(ΔUbL)Rad₂₃-HA). We found that^(ΔUbL)Rad23-HA was stable in actively growing cells (FIG. 9A),displaying a half-life that exceeded 10 hours during exponential growth.Significantly, ^(ΔUbL)Rad23-HA failed to complement the UV sensitivityof rad23Δ (Watkins et al. 1993), suggesting that UbL^(R23) may have aproteolytic function in DNA repair.

[0090] The Ubiquitin-like Domain is an Autonomous Degradation Signal

[0091] The ability of UbL^(R23) to promote the degradation of a reporterprotein was tested by linking it to β-galactosidase (UbL^(R23)-βgal).The data illustrate that UbL^(R23)-βgal is stable in stationary-phasebut exceedingly unstable during active growth (FIG. 9A and FIG. 9B),intensifying the degradation pattern of Rad23-HA (FIG. 7A). Longover-exposures of the autoradiograms revealed a low level of UbLR²³-βgalin the 0 min sample in logarithmically growing cells, and quantitativeβ-galactosidase activity measurements confirmed these findings (data notshown). These results demonstrate that UbL^(R23) is both necessary andsufficient for the targeting and degradation of Rad23-HA, and ispredicted to contain amino acid residues that are recognized byproteolytic factors. Furthermore, UbL^(R23) contains sequences that aresensitive to regulatory signals because UbL^(R23)-βgal mimicked theregulated degradation of Rad23-HA. UbL^(R23)-βgal migrated as a set of 3closely spaced electrophoretic bands. It is not known if these bandscorrespond to multiubiquitination or other modifications ofUbL^(R23)-gal.

The Ubiguitin Fusion Degradation (UFD) Pathway is Involved in theDegradation of Rad23-HA

[0092] The placement of ubiquitin on the N-terminus of a protein such asβ-galactosidase (Ub-P-βgal), can promote degradation by the Ubc4ubiquitin-conjugating enzyme (Bachmair et al., 1986). Ubc4 assembles amultiubiquitin chain at a conserved lysine in the Ub extension ofUb-P-βgal (Johnson et al., 1992). Since the lysine residues which serveas attachment sites for the formation of a multiubiquitin chain areconserved between Ub and UbL^(R23), we predicted that Rad23-HA mightalso be targeted by Ubc4. Ubc5 encodes another ubiquitin-conjugatingenzyme which is approximately 90% identical to Ubc4 and is believed tohave overlapping substrate specificity (Seufert and Jentsch, 1990).Rad23-HA stability was examined in ubc4Δ ubc5Δ. In these cells,degradation of the protein was unaffected (FIG. 6A) compared to thewildtype strain (data not shown). These findings show that this class ofE2 enzymes does not target Rad23-HA for degradation.

[0093] In a search for factors that affect the degradation of Ub-P-βgalJohnson, et al. performed a genetic screen and identified a class ofmutants (termed the UFD pathway- for ubiquitin fusion degradationpathway) that differentially affected Ub-P-βgal stability. Johnson etal. determined that UFDS was the only UFD pathway gene that was alsorequired for the degradation of N-end rule substrates, which aredistinct from Ub-P-βgal. While N-end rule substrates are ubiquitinatedby Ubc2 and Ubr1, Ub-P-βgal is ubiquitinated by Ubc4. The possibilitythat the ufd mutants might affect the stability of Rad23-HA wasexamined. Pulse-chase measurements showed that Rad23-HA was stronglystabilized in ufd5Δ (FIG. 10E), but not in ufd1-ufd4 (FIGS. 10B-10D).Multiubiquitinated derivatives of Ub-P-βgal were detected in ufd5Δ,while Rad23-HA accumulated as an apparently unmodified protein. Althoughthe biochemical activity of Ufd5 is unknown, these results demonstratethat the channeling of substrates to the proteasome can follow diverseroutes. This observation is also supported by our finding that differentproteasome mutants have distinct effects on Rad23-HA stability (seebelow).

Proteasome Dependent Degradation of Rad23-HA

[0094] Substrates of the Ub system are generally degraded by the 26Sproteasome, an evolutionarily conserved structure of >2×10⁶ Daltons. Itwas recently reported that a yeast pheromone-specific receptor, Ste2, isubiquitinated but degraded in the vacuole in a proteasome-independentmanner (Hicke and Riezman 1996). In contrast ornithine decarboxylase(ODC) is degraded by the 26S proteasome, although it is notubiquitinated (Tokunage et al. 1994). Given these exceptions, to thegenerally accepted model for targeting and degradation of ubiquitinatedsubstrates, we measured the stability of Rad23-HA in yeast strainsbearing mutations in either proteasome subunits or vacuolar proteases,to determine if its degradation involved the ubiquitin/proteasomepathway. Cim5 is an ATPase subunit of the 19S regulatory complex of the26S proteasome and is required for the degradation of Ub-P-βgal(Ghislain et al. 1993). The stability of Rad23-HA in exponential-stagecim5-1 cells was measured and the results show that it was very stable(t_(½)>10 hrs, FIG. 11B). Pre1 and Pre2 are subunits of the 20Scatalytic core of the 26S proteasome, and mutants are sporulationdefective and stress-sensitive. In agreement with the results observedin cim5-1, we found that Rad23-HA was stabilized in actively growingpre1-1 pre2-2 cells (t_(½) ⁻1 hr, FIG. 11A). In contrast the degradationRad23-HA was unaffected in pep4Δ prb1-Δ1, which is defective in vacuolarproteolysis ((Hicke and Riezman 1996), FIG. 11E. Thus, it appears thatRad23-HA degradation requires the 26S proteasome.

[0095] The very rapid degradation of Rad23-HA precluded detection ofmultiubiquitin intermediates. Ubiquitinated Rad23 was previouslydetected (Watkins et al. 1993), suggesting that Rad23-HA degradation isubiquitin-mediated. Mcb1 is a yeast counterpart of the human S5a proteinwhich encodes a multiubiquitin-chain binding protein of the 26Sproteasome. The stability of Rad23-HA was tested in mcb1Δ and resultsdemonstrated that it continued to be degraded rapidly (FIG. 11D). Sincemcb1Δ stabilizes only a subset of ubiquitinated substrates in yeastcells it is possible that other multiubiquitin-chain binding proteinscan mediate Rad23-HA degradation. The stability of Rad23-HA in doa4Δ-1,an isopeptidase associated with the 26S proteasome, was also measuredsince many substrates of the ubiquitin system are stabilized in thismutant. Surprisingly, Rad23-HA continued to be degraded in doa4Δ-1 (FIG.11C). These results demonstrate that substrates of the ubiquitin systemcan follow diverse routes into the proteasome.

Rad23 Interacts with Other Components of the Proteasome

[0096] Rad23 was linked to lexa and interacting factors were sought bythe 2-hybrid method (Paetkau et al. 1994). We identified Irt1(interaction with Rad23), a protein whose C-terminal domain displayssignificant homology with the 26S subunit Yta6 (FIG. 8A, Schnall et al.1994) and Cim3 and Cim5. See FIG. 12A. The large N-terminal domain ofIrt1 is not similar to any known polypeptide sequence. The degradationof Rad23-HA was unaffected in irt1Δ (data not shown), indicating thatthe interaction between Rad23 and Irt1 is likely to be of a regulatorynature.

[0097] To further characterize the interaction between Rad23 and Irt1several deletion derivatives of Irt1 were constructed and tested fortheir ability to interact with Rad23 (FIG. 12B). Full-length Irt1 (897amino acids) as well as three C-terminal deletion variants, comprisingresidues 1-567, 1-243 and 1-172, were tested in the 2-hybrid system.Irt1¹⁻⁵⁶⁷ lacks the highly conserved ATPase domain located in theC-terminus, while the larger truncations removed additional residues ofunknown function.

EXAMPLE III Ubiquitin-like Sequences are Proteasome Interacting Domains

[0098] A family of proteins that contain ubiquitin-like sequences(UbL's) has been identified in diverse organisms (Garrett et al., 1995;Shen et al., 1996). Some UbL's are post-translationally conjugated toother proteins in a mechanism similar to that described forubiquitin-conjugation (Johnson et al., 1997; Mahajan et al., 1997).However, a distinct class of UbL's are retained in the originaltranslational product and not conjugated to other proteins (Watkins etal., 1993). The proteins to which these UbL's are fused share little incommon and offer no obvious clues to their biological functions.Furthermore, the effect of a UbL on the activities of the protein towhich it is linked is unknown. Although UbL's display no more than20-30% identity to the amino acid sequence of ubiquitin, their3-dimensional structures are predicted to be highly similar (van derSpek et al., 1996). The two proteins in yeast that contain N-terminalubiquitin-like domains were reported to be stable (Biggins et al.,1996). However, we have determined that Rad23 is ubiquitinated anddegraded during the G1/S-phase transition of the cell-cycle. The fusionof ubiquitin to the N-terminus of β-galactosidase (Ub-Pro-βgal) has alsobeen shown to cause rapid degradation by the ubiquitin pathway (Johnsonet al., 1995; Bachmair et al., 1986).

[0099] Dsk2 is another yeast protein that contains a ubiquitin-likedomain (UbL^(DSK)), and deletion of both genes (rad23 dsk2) causes atemperature sensitive growth defect (15), suggesting that theiractivities converge at some unknown biochemical level.

[0100] To examine if Rad23 associated with proteolytic factors we linkedRad23 and UbL^(R23) to GST and found that both GST-Rad23 andGST-UbL^(R23) formed stable interactions with the 26S proteasome. SeeExample I. The data presented herein demonstrate thatproteasome-interaction is a feature shared by other members of thefamily of ubiquitin-like proteins, and indicate that UbL containingproteins mediate proteolytic functions. UbL's and UbL-containingproteins have been implicated in many biological pathways including DNArepair (Watkins et al., 1993), spindle pole-body duplication (Biggins etal., 1996), transcription elongation (Garrett et al., 1995), von HippleLandau syndrome (Kibel et al., 1995) and nuclear/RNA transport (Mahajanet al., 1997). The best characterized among these proteins is yeastRad23.

[0101] The findings presented indicate that UbL/proteasome interactionis regulated. The UbL from yeast Dsk2 (UbLDSK) interacts with theproteasome preferentially in actively growing cells. Overexpression ofUbL^(R23) inhibits the degradation of specific substrates of theubiquitin pathway perhaps by saturating the proteasome targetingpathway. Significantly, our results show that UbL-linked proteinsinteract with the proteasome without prior attachment to amultiubiquitin chain, defining a novel mechanism for targeting proteinsto the proteasome.

Materials and Methods for Example III Strains and Extracts

[0102] The yeast strains used in these studies were derived fromJD47-13C; MATahis3-Δ200trp1-Δ63 lys2-801 ura3-52 leu2-2, 112 (J.Dohmen). Rad23 deletion (CSY85; rad23Δ::URA3) was made in JD47-13C usingpDG28. Extracts for immunoprecipitations and affinity purifiedpurification were described previously. (Schauber et al., 1998).

Reagents

[0103] Proteasome inhibitors were obtained from Calbiochem,glutahione-Sepharose from Pharmacia, anti-ubiquitin antibodies fromSigma, and anti-βgalactosidase antibodies from Promega.

Plasmids and Constructs

[0104] UBL's and CIM5 were amplified by PCR with oligonucleotidescontaining a 5′ NcoI and 3′ KpnI restriction site and ligated intosimilarly treated pCBGST1 (Schauber et al., 1998). The expression of theproteins was induced with 0.15 mM CUSO₄. Plasmids encoding Pre-1-FLAGand Sen3-HA were provided by J. Dohmen and M. Hochstrasser.

[0105] UbL's are proteasome-interacting sequences. The ubiquitin-likedomains of yeast Rad23 and Dsk2, and human HHR23A and -B, were linked tothe C-terminus of glutathione S-transferase (GST), and expressed inyeast. Extracts were incubated with glutathione-Sepharose, and boundproteins separated in a SDS-polyacrylamide gel, transferred tonitrocellulose and analyzed by immunological methods. The blot wasincubated with Cim3 and Cim5 antibodies, which recognize subunits of the26S proteasome, and a strong interaction was detected in the beadscontaining GST-UbL^(R23). See FIG. 13A, lane 2. UbL^(R23) /proteasomeinteraction was resistant to 1M NaCl, and treatment with detergentsincluding 1% Triton X-100, 0.5% NP40 and 0.1% SDS (data not shown).Ubiquitin (GST-Ub) did not interact appreciably with the 26S proteasome(FIG. 13A, lane 3), supporting the idea that ubiquitin is recognized bythe proteasome only when it is assembled into a multiubiquitin chain(Chau et al., 1989). In contrast, the UbL may have evolved tospecifically interact with the proteasome without prior attachment to amultiubiquitin chain. Weaker interactions were detected withGST-UbL^(DSK) and GST-UbL^(HRB) (lanes 4 and 6), but not withGST-Ub^(HRA) (lane 5). To examine the possibility that GST-UBL^(HRA) andGST-UbL^(HRB) might interact more favourably with human proteins weincubated Hela cell S100 extracts with GST-UbL^(HRA) and GST-UbL^(HRB).The interacting proteins were analyzed in a western blot with Cim5antibodies which crossreact with Mss1, a human counterpart of yeast Cim5(Ghislain et al., 1993). Mss1 was detected in GST-UbL^(HRA) andGST-UbL^(HRB) beads (FIG. 14A, lanes 2 and 3), but not GST (lane 1). Acontrol lane containing GST-UbL^(R23) interacting proteins showed thatthe antibody reaction against Cim5 was efficient (FIG. 14A, lane 4). Inagreement with these findings we found that cells expressing Sen3-HA, anon-ATPase 19S subunit (DeMarini et al., 1995), also interacted withGST-UbL^(R23) but not GST (see FIG. 14A).

[0106] Consistent with these results, we detected Pre1-FLAG (anepitope-tagged 20S subunit) in beads containing GST-UbL^(R23),GST-UbL^(DSK), and GST-UbL^(HRB) (FIG. 13B). These results show thatseveral different subunits of the 19S and 20S components of the 26Sproteasome can be detected in a complex that interacts withubiquitin-like domains. We conclude that a common biochemical propertyof a UbL is its interaction with catalytically active 26S proteasome.

[0107] UbL's interact with the 198 regulatory component of the 26Sproteasome. Based on the activities associated with UbL^(R23), theproteasome interacting-subunit could be located in either the 19S or 20Scomplexes. To examine the interaction with the 19S regulatory complexGST-UbL^(HRA) and GST-UbL^(HRB) were incubated with approximately 5 μg19S/PA700 (a gift from Dr. G. DeMartino, Univ. of Texas, Dallas, Tex.),for 10 hr at 4° C. Bound proteins were resolved in SDS-PAGE and examinedby silver staining. A significant fraction of the input protein wasdetected in the beads containing UbL^(HRA) and UbL^(HRB) (FIG. 14B,lanes 2 and 3). The profile of 19S/PA700 subunits that boundGST-UbL^(HRA) and GST-UbL^(HRB) was similar demonstrating that theentire complex, rather than specific subunits, interacts with the UbL.UbL^(HRB), but not UbL^(HRA), showed detectable interaction with yeastproteasomes, although both chimeras bound human proteasome (FIG. 14).This variance in interaction may result from subtle differences in theirsequences, which might offer clues to the residues that are importantfor proteasome binding.

[0108] A novel mechanism is involved in UbL^(R23)/proteasomeinteraction. Substrates of the ubiquitin system are covalently linked toa multiubiquitin chain prior to recognition by the 26S proteasome. In asearch for multiubiquitin-chain binding proteins van Nocker et al.,identified Mcb1, which is a component of the 19S regulatory complex ofthe proteasome (van Nocker et al., 1996). Since UbL's interact with theproteasome through the 19S complex (FIG. 14B), we investigated ifUbL^(R23) could interact with the proteasome in mcb1Δ. We purifiedGST-UbL^(R23) from mcb1Δ and found that it co-precipitated Cim5 andCim3, demonstrating that its interaction with the proteasome is notmediated by Mcb1. This result proves that there are alternate ways forsubstrates and regulators to interact with the proteasome, and isconsistent with studies which showed that some substrates of theubiquitin system are efficiently degraded in mcb1Δ (van Nocker et al.,1996).

[0109] UbL^(R23) can target heterologous proteins to the proteasome.UbL^(R23) was linked to the N-terminus of β-galactosidase(UbL^(R23)-βgal) and Ura3-HA (UbL^(R23) -Ura3-HA), and the plasmids weretransformed into a yeast strain expressing GST-Cim5. Extracts wereincubated with anti-β-galactosidase or anti-HA antibodies, andimmunoprecipated protein recovered on Protein-A Sepharose beads,resolved in SDS-PAGE and transferred to nitrocellulose. Thenitrocellulose filter was incubated with anti-GST antibodies, and theposition of full-length GST-Cim5 from a control extract, is indicated bythe arrow (FIG. 15, lane 1). We found that GST-Cim5 was highlysusceptible to proteolysis (as indicated by the large number of smallerfragments). Extracts containing GST-Cim5 and UbL^(R23)-βgal wereincubated with anti-β-galactosidase, and a strong reaction againstGST-Cim5 was detected in the immunoprecipitates (FIG. 15, lane 2).Interestingly, the degradation products of GST-Cim5 (lane 1), were notseen in lane 2 suggesting that only intact GST-Cim5 is incorporated intothe proteasome. Extracts containing only GST-Cim5 were also incubatedwith anti-βgal antibodies and resolved on the gel. As expected, GST-Cim5was not precipitated in this reaction (FIG. 15, lane 3). To extend thesefindings further we examined if UbL^(R23)-Ura3-HA could also selectivelyprecipitate GST-Cim5. A band consistent with GST-Cim5 was detected (lane4), and as observed in lane 3 only intact GST-Cim5 protein wasprecipitated with UbL^(R23)-Ura3-HA. An extract containing only GST-Cim5was reacted with anti-HA antibodies and GST-Cim5 was not precipitated(lane 5). We conclude that UbL^(R23) is an autonomous sequence that cantarget unrelated proteins to the proteasome.

[0110] UbL/proteasome interaction can be regulated. We found thatUbL^(DSK) forms a weak association with the proteasome. The function ofDsk2 is expected to be confined to actively growing cells because it isrequired for spindle pole-body duplication. Our preliminary studiesindicate that UbL^(DSK) interacts more favorably with the proteasome inactively growing cells. It remains to be determined how the naturalC-terminal extension regulates UbL/proteasome interaction. We havereported in Example I that specific alleles of Rad23 are rapidlydegraded by the ubiquitin/proteasome pathway, in a mechanism thatrequires UbL^(R23). Since these Rad23 variants are degraded only inactively growing cells, it appears that UbL^(R23) /proteasomeinteraction may also be regulated.

[0111] UbL^(R23) interferes with proteasome function. The high affinityinteraction between UbL^(R23) and the 26S proteasome suggested that itmight affect the degradation of substrates of the ubiquitin system. Weexamined the stability of substrates of the N-end rule (Arg-βgal andLeu-βgal) and UFD pathways (Ub-Pro-βgal), by measuring β-galactosidaseactivity of test substrates. The levels of Leu-βgal and Ub-Pro-βgal were2-3 fold higher in cells expressing GST-UbL³, than in the GST control.In contrast, the activity in cells expressing Met-βgal and Arg-βgal wasunchanged (data not shown). To confirm these results we measured the invivo half-lives of test substrates by pulse-chase analysis. We foundthat Leu-βgal and Ub-Pro-βgal were moderately stabilized in cellsexpressing GST-UbL^(R23) (FIG. 16B), as compared to GST (FIG. 16A).These results are in agreement with the β-galactosidase activitymeasurements. Significantly, ubiquitinated derivatives of Leu-βgal andUb-Pro-βgal accumulated in cells expressing GST-UbL^(R23), indicatingthat UbL^(R23) interferes with a post-targeting step in substratedegradation. This effect is most easily seen in Leu-βgal levels at the 0time-point. These results suggest that UbL^(R23) interaction with theproteasome can block, or otherwise interfere with, the access ofspecific substrates to the proteasome. The alternate possibility thatGST-UbL^(R23) increased ubiquitin-conjugation is considered less likelybecause the rate of Leu-βgal degradation was reduced, and not increasedas would be expected if it was more efficiently targeted. In contrast tothe stabilization of Leu-βgal, Arg-βgal remained extremely unstable instrains expressing either GST or GST-UbL^(R23). This result suggeststhat the fate of Type I (Arg-βgal) and Type II (Leu-βgal) substrates ofthe N-end rule pathway may diverge following their conjugation toubiquitin by the targeting components Ubr1/Ubc2).

[0112] The function of ubiquitin-like domains (UbL) was previouslyunknown. We report here that the UbL is a cis-acting signal that cantranslocate UbL-linked proteins to the proteasome. In addition to theRad23 proteins and Dsk2, we also examined the interaction betweenElongin-B and the proteasome. Consistent with the findings shown in FIG.13, we detected an interaction with Cim5 (data not shown). Elongin B isa UbL-containing protein that forms a heterotrimeric complex whichmodulates transcription by RNA Pol II. We have also reported elsewherethat UbL^(R23) can function as a portable degradation signal, when fusedto the N-terminus of β-galactosidase (UbL^(R23)-βgal). Although thebiological significance of UbL/proteasome interaction is unknown, wesuggest that UbL's can be either substrates or regulators of theproteasome. There also exists an interesting possibility that aUbL-linked protein can promote the degradation of other proteins intrans, by binding and transporting them to the proteasome. A particularadvantage of this mechanism for proteasome targeting is that anelaborate ubiquitin-dependent apparatus is dispensed with, and the invivo levels of a substrate could be regulated by the concentration ofits cognate UbL-containing partner, and its affinity for the proteasome.A precedent for this mechanism is noted by the (ubiquitin-independent)antizyme-mediated degradation of ornithine decarboxylase by theproteasome (Murakami, et al., 1992). A potential target forRad23-mediated degradation could be Rad4, to which it binds with highaffinity. Rad23 and Rad4 are both important for the assembly of thenucleotide excision repair complex, and genetic and biochemical studieshave implicated a regulatory role for Rad23. We have shown that Rad23and Rad4 can be purified in a complex with the proteasome, although itremains to be determined if Rad23 influences Rad4 stability. TheRad23-mediated link between DNA repair and protein degradation maydefine a mechanism to recycle the repair complex, or to facilitaterecovery after the completion of DNA repair.

[0113] Of the four UbL's present in yeast, only Smt3 and Rub1 areconjugated post-translationally to other proteins. A mammaliancounterpart of Smt3 (SUMO) is covalently linked to RanGAP1, althoughthis modification does not appear to promote degradation. However, it ispossible that only a small fraction of RanGAP1 is post-translationallymodified, and its SUMO-mediated turnover may be masked by the largefraction of unmodified RanGAP1. We showed in FIG. 13 that mono-ubiquitin(GST-Ub) failed to interact with the proteasome. Since ubiquitininteracts with the proteasome only when it is assembled into amultiubiquitin chain, we propose that substrate-linked Smt3 andsubstrate-linked Rubl might also be targeted to the proteasome. Thisidea can be tested once the physiological targets of Smt3 and Rub1 areidentified.

[0114] UbL-containing proteins may prevent the degradation of otherproteins by blocking their access to proteolytic factors. For instance,GST-UbL^(R23) interaction with the proteasome inhibited the degradationof specific substrates of the ubiquitin pathway (FIG. 16). We found thatUbL^(R23) stabilized Leu-βgal but not Arg-βgal, which are distinctsubstrates of the N-end rule pathway.

[0115] UbL^(R23) also stabilized Ub-Pro-βgal, a substrate of the UFDpathway. Pulse-chase experiments suggested that inhibition ofdegradation occured at a post-targeting step because multi-ubiquitinatedderivatives of Leu-βgal and Ub-Pro-βgal accumulated in the presence ofGST-UbL³.

[0116] Rad23 and Dsk2 are the only yeast proteins that retainubiquitin-like domains in the mature proteins. Rad23 is required fornucleotide excision repair, while Dsk2 is involved in spindle pole-body(SPB) duplication. Deletion of both genes (rad23 dsk2) causes atemperature sensitive growth defect indicating that the biochemicalactivities of Rad23 and Dsk2 intersect, possibly at the level of the 26Sproteasome. UbL^(R23), but not UbL^(DSK), interferes with thedegradation of specific test proteins. We suggest that substrates of theN-end rule and UFD pathway may be channeled to a specific proteasomeisoform that is recognized only by UbL^(R23) Interestingly,UbL^(DSK)/proteasome interaction is enhanced in actively growing cells,also suggesting that compositionally distinct types of proteasomes mayregulate UbL interactors. This observation is consistent with a previousstudy which showed that specific 20S proteasome subunits are replacedfollowing γ-interferon treatment in mammalian cells (Gaczynska, et al.,1993).

EXAMPLE IV Enhanced Thermostability of Rad23 and Use of the UbL^(R23)Domain to Confer Thermostability on Fusion Protects

[0117] Rad23 was purified to homogeneity from bacteria and subjected tostructural analysis by circular dichroism (CD-spectra). The analysis wasdone along with other proteins unrelated to this work. The CD-spectrarevealed that Rad23 is a typical globular protein, which is highlysoluble and contains substantial a-helical character. See FIG. 17.However, when the thermal stability of the protein was analyzed the datarevealed that it did not display the cooperative melting profiletypically observed for globular proteins. See FIG. 18. Indeed a meltingtransition was not detected even when Rad23 was heated to excess of 90°C. Consistent with this result when the protein sample was returned to23° C. it continued to display CD-spectra consistent with a well-foldedglobular and soluble protein. In contrast, other proteins that wereanalyzed at the same time displayed the expected cooperativedenaturation at 52° C. indicating that the experimental conditions andthe function of the instrument were normal.

[0118] The results obtained indicate that the UbL is a cis-acting,temperature stabilizer. As described in the previous examples, UbL^(R23)has been fused to β-galatosidase. Like Rad23, this fusion protein can beheated without loss of secondary structure. Additionally, exposure tohigh temperatures did not inactivate the enzymatic portion of the fusionprotein.

[0119] This observation indicates that the UbL has broad applications inthe generation of fusion proteins having enhanced thermostability.

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[0197] While certain of the preferred embodiments of the presentinvention have been described and specifically exemplified above, it isnot intended that the invention be limited to such embodiments. Variousmodifications may be made thereto without departing from the scope andspirit of the present invention, as set forth in the following claims.

What is claimed is:
 1. A method for rapid and efficient purification ofproteasomes from cells comprising: a) immobilizing an amino acidsequence having a UbL domain to a solid support; b) exposing saidimmobilized UbL domain to a cell lysate; c) eluting non-specificallybound proteins; and d) eluting said proteasome from said solid support,thereby purifying said proteasome from said cell lysate.
 2. A method asclaimed in claim 1, wherein said UbL domain has the amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.
 3. A method as claimed inclaim 1, wherein said UbL domain and said cell lysate are isolated fromthe same species.
 4. A kit for the rapid purification of proteasomesfrom a cell lysate, said kit containing: a UbL domain affixed to a solidsupport, one or more containers, a wash solution and an elution buffer.5. A kit as claimed in claim 4, further comprising a solution useful inperforming a purification method of the invention, selected from thegroup consisting of saline, buffer, diluent, and frozen cell extract. 6.A DNA construct encoding a fusion protein for assessing theproliferative potential of malignant cells comprising: a) a firstnucleic acid sequence encoding a promoter element; and b) a secondnucleic acid sequence encoding a UbL domain operably linked to a thirdnucleic acid sequence encoding a reporter gene, expression of said UbLdomain and said reporter gene being regulated by said promoter.
 7. A DNAconstruct as claimed in claim 6, said construct being inserted into avector.
 8. A DNA construct according to claim 6, wherein said secondnucleic acid encodes a UbL domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, and SEQ ID NO:12.
 9. A DNA construct according toclaim 6, wherein said reporter gene is selected from the group of genesconsisting of βgalatosidase, URA3, luciferase, mammalian chloramphenicoltransacetylase (CAT) gene, and green fluorescent protein (GFP) gene. 10.A method for assessing the proliferative potential of malignant cells,comprising: a) introducing into a target cell a DNA construct encoding afusion protein, said fusion protein comprising a UbL domain operablylinked to a reporter molecule; and b) assessing the half-life of saidfusion gene, a short half-life being indicative of a rapidly growingcell and a longer half-life being indicative of a quiescent cell.
 11. Amethod as claimed in claim 10 wherein said UbL domain has an amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.
 12. A method asclaimed in claim 10, wherein said reporter gene is selected from thegroup of genes consisting of βgalatosidase, URA3, luciferase, mammalianchloramphenicol transacetylase (CAT) gene, and green fluorescent protein(GFP) gene. consisting of.
 13. A DNA construct encoding a thermostablefusion protein, comprising a first nucleic acid sequence encoding a UbLdomain operably linked to a second nucleic acid sequence encoding aprotein of interest.
 14. A DNA construct as claimed in claim 11, whereinsaid first nucleic acid sequence encodes a UbL domain having an aminoacid sequence selected from the group consisting of SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.
 15. A DNAconstruct as claimed in claim 11, wherein said second nucleic acidmolecule encodes a polymerase enzyme.
 16. A DNA construct encoding afusion protein for selecting for drug resistance in malignant cellscomprising: a) a first nucleic acid sequence encoding a promoterelement; and b) a second nucleic acid sequence encoding a UbL domainoperably linked to a third nucleic acid sequence encoding a selectablemarker gene, expression of said UbL domain and said selectable markergene being regulated by said promoter.
 17. A DNA construct as claimed inclaim 16, wherein said second nucleic acid gene encodes a UbL domainhaving a sequence selected from the group consisting of SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.
 18. ADNA construct as claimed in claim 16, wherein said selectable markergene is selected from the group consisting of the neomycin gene, orantibiotic resistance genes.